Microbiology Lab

Exercise 10: Smear Preparation

Exercise 11: Simple Staining

Ex 12: Negative Staining

REVIEW LAB RESULTS FROM LAST WEEK

• Ex 8: Aseptic Technique
• Did you successfully get bacteria growing in your broth and slant?

• Ex 6: The Ubiquity of Bacteria
• Which plate did you expect to have the worst contamination?
• Which plate actually had the worst contamination?
• What surprised you the most about the experiment?

Ex 10: Smear Preparation

1. Every student will prepare 2 slides of E. coli (1 from the slant & 1 from the broth started by students last week) for use in the simple staining lab.
2. Procedure
1. Gather supplies

i. Bacterial cultures (1 slant & 1 broth of E. coli)

ii. Clean microscope slides

iii. Inoculating loop

iv. Water bottle

v. Sharpie pen

vi. Bunsen burner & striker

2. Label slide

i. Label a short edge of the slide with the bacteria, your initials, & date

ii. Draw a target circle about ½” wide

iii. Turn your slide over so you work on the unlabeled side

3. Aseptically collect a sample of your bacteria

i. When you are using broth, transfer two loopfuls of bacteria to your slide

ii. When you are suing solid media, place 2 loopfuls of water on the slide, then transfer a SMALL amount of organisms to your slide

4. Spread your organisms out over the entire area of your target circle
5. Let the smear air dry
6. Heat fix the slide by passing it through the flame of a Bunsen burner until the slide is very warm to the touch

Ex 11: Simple Staining

· Look at the view of Corynebacterium diphtheriae at

www.mf.uni-lj.si/imi/images/preparati/mikrobi/coryn01.jpg

o I went to google & searched “Corynebacterium diphtheriae images”

o This slide was prepared by…

§ Preparing a smear.

§ Staining with methylene blue for one minute

o Note the following in this view…

§ Pleomorphism – irregularity of form

· C. diphtheriae is a bacillus, but may look like a rod, club-shaped, spermlike, or needle-shaped

§ Metachromatic granules

§ Palisade arrangement – a “picket fence” arrangement

· Prepare a simple stain of Escherichia coli

o Use your prepared smear of E. coli

o Stain with methylene blue for one minute

o Rinse with water

o Blot with bibulous paper

o Observe under oil immersion lens

· What is the morphology and arrangement of E. coli?

Gram Stain of E. coli

Ex 12: Negative Staining of Staphylococcu aureus and Bacillus megaterium

· Negative stains – stains that don’t penetrate the bacterial cell wall, but cause the background slide to appear dark while the bacteria remain colorless

· Prepare 2 negative stain slides: 1 each of S. aureus and B. megaterium

o Label the slide with the bacteria’s name, your initials, & date

o Place a loopful of India ink in the center of your slide

o Aseptically collect a loopful of bacteria and transfer them to the slide with the India ink

o spread the organisms out over an area of about ¾”

o Allow to air dry

o View under oil immersion

· What is the morphology & arrangement of S. aureus?

· What is the morphology & arrangement of B. megaterium?

Negative stain of B. megaterium (note the endospores that don’t take up the stain)	Gram stain of S. aureus